Insulin production consists of a series of interdependent events involving movement of precursors from one organelle to the next and their proteolytic conversion. The underlying hypothesis of this Project is that structural domains of insulin and its precursors are implicated in each of these steps. Other domains will be equally important once insulin is released from the B-cell, being responsible for biological activity and, ultimately, degradation by target tissues. The longterm goals of this study are to: a) characterize each step in insulin production and pinpoint defects responsible for abnormal insulin production in given diabetic states; b) study the interplay between human and mouse insulins in transgenic mice in order to understand better the impact of bioartificial insulin delivery on B-cell function. The specific aims for the next 3 Year Period are: 1. How is proinsulin transported from the RER to the cis-Golgi? The effects of inhibitors on localization (immunocytochemistry) and chemistry of proinsulin in rat islet B-cells will be evaluated. Brefeldin A inhibits RER-Golgi transfer but allows the reverse movement of molecules (regurgitation from the Golgi). NEM (N-ethylmaleimide) prevents fusion of transport vesicles with their target organelle. Cyproheptadine leads to accumulation of material in dilated cisternae of the RER. 2. Which proinsulin domains are involved in targeting from the trans-Golgi to granules and in recognition by the conversion endoproteases? Proinsulin conversion/release will be studied in transfected AtT20 (pituitary corticotroph) cells. Studying expression of mutant genes will show whether the altered domain is involved in targeting or conversion. 3. Can proinsulin be converted (or partially converted) if released via the constitution pathway? FAO (hepatoma) cells will be transfected with the proinsulin gene. Since these cells do not express the regulated pathway, all proinsulin must be handled by the constitutive pathway. HPLC analysis of products synthesized and released by transfected FAO cells will show whether any conversion arises in this pathway. 4. Why is rat proinsulin I converted to insulin more rapidly than proinsulin II, and is there any difference in the biological activity of the two rat insulins? The rate of conversion of proinsulin to conversion intermediates (split proinsulins) and of intermediates to insulin will be studied in rat islets. The affinity of the rat insulin receptor for the two insulins will be measured, and the kinetics of receptor-mediated degradation followed. 5. Is newly synthesized proinsulin/insulin really released in preference to older, stored, insulin, or is the phenomenon merely a reflection of B- cell heterogeneity? B-cells will be separated from non-B-cells by flow cytometry and further sorted into metabolically active and inactive subpopulations based upon NAD(P)H autofluorescence. Rates of release of new (labeled) and old insulin will then be followed from the two subpopulations. 6. How is human insulin synthesis regulated in transgenic mouse B-cells, and what is its impact on endogenous insulin production and metabolism? Rates of synthesis and release of human and mouse insulins, as well as their biological activity, will be compared in transgenic mice expressing human insulin in their B-cells.